Kit Contents:

Component	Specification	Concentration (total volume)
RARE Reporter	A mixture of inducible RAR-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).	(100 ng/µl; 500 µl)*
Negative control	A mixture of non-inducible firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).	(100 ng/µl; 500 µl)
Positive control	A mixture of constitutively expressing GFP, constitutively expressing firefly luciferase, and constitutively expressing Renilla luciferase constructs (40:1:1).	(100 ng/µl; 250 µl)

* Supplied material provides sufficient reporter for 500 assays, using recommended 96-well plate transfection protocol. The number of assays per kit is a function of the assay plate format used (refer to Cignal Reporter Assay User Manual).

Storage Conditions: The Cignal reporter assay constructs are shipped ambient. Store all tubes at -20  ºC.

1. Dilute transfection-ready reporter, negative control, and positive control construct formulations.
2. Dilute relevant test nucleic acids (siRNA, shRNA, miRNA, expression vector).
3. Prepare appropriate combinations of reporter constructs, controls, and test nucleic acids.
4. Transfect plasmid mixtures separately into replicate wells of your cell line of interest using an optimized transfection procedure for the cell line under study.
5. If applicable, 16 to 24 hours post-transfection, treat the transfected cells with test proteins, peptides, or compounds of interest.
6. Two (2) to three (3) days post-transfection, assay the activities of the signaling pathways under study, utilizing the dual luciferase assay.

The Cignal Reporter Assays include pre-formulated, transfection-ready reporter, negative control, and positive control. The transcription factor reporter and negative control are transfected and subjected to experimental treatments, in parallel. Dual-luciferase results are calculated for each transfectant. The impact of the experimental treatments is determined by comparing the normalized luciferase activities of the reporter to the identically treated negative control, across the complete treatment regimen. The positive control serves as a control for transfection efficiency, by monitoring GFP expression, as well as a positive control for both the firefly and Renilla luciferase assays.

General performance

Average maximum response rate = 14
Average coefficient of variation (CV%) = 10%

Excellent signal to noise ratio

Cignal RARE reporter assay can measure increase in retinoic acid receptor pathway activity: CHO-K1 cells were transfected with RARE reporter, negative control and positive control (for transfection protocol refer our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 1% charcoal stripped FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 24 hours of transfection the cells were treated with 1µM all trans-rectinoic acid (ATRA) for 6 hours. Dual Luciferase assay was performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated.

Retinoic Acid, a lipophilic molecule and a metabolite of Vitamin-A (all-trans-Retinol), affects gene transcription and modulates a wide variety of biological processes like Cell Proliferation, Differentiation, including Apoptosis. Retinoic Acid mediated gene transcription depends on the rate of transport of Retinoic Acid to target cells and the timing of exposure of Retinoic Acid to RARs (Retinoic Acid Receptors) in the target tissues. The all-trans-Retinoic Acid, the Carboxylic Acid form of Vitamin-A is of biological significance since it has high circulating levels than other isomers of Retinoic Acid. The targets of all-trans-Retinoic Acid and RARs include a multitude of Structural genes, Oncogenes, Transcription Factors and Cytokines. Although biologically active ligands for the RARs also include 9-cis-Retinoic Acid among others, yet circulating levels of 9-cis-Retinoic Acid are much lower than those of all-trans-Retinoic Acid and  the physiological significance of the isomerization all-trans-Retinoic Acid to 9-cis-Retinoic Acid and vice versa  is yet to be ascertained (Ref.1). The all-trans-Retinoic acid is predominant under most physiological situations and explains all of the biological effects of Vitamin-A. The RARs are encoded by three separate genes with multiple isoforms-Alpha, Beta and Gamma, which are generated by alternative promoters and differential splicing. Like all NRs (Nuclear Receptors) RARs also have a conserved modular structure consisting of an AF-1 or A/B (Amino-Terminal Activating Factor-1 Transcriptional Activation) Domain; a zinc-finger DBD or C (DNA-Binding Domain); a CoR or D (Hinge/Corepressor Binding) Domain; a LBD or AF-2 or E (Ligand-Binding/Transcriptional Activation) Domain; and a variable F (Carboxyl-Terminal) Domain. In general, the RARs contain six regions from A-F. The DBD binds to the RARE (Retinoic Acid Response Element) region in the DNA. The RAREs consists of a DRs (Direct Repeats) of AGG/TTCA motif with a spacer region of (n)25. Vitamin-A in the liver is converted to all-trans-Retinoic Acid, diffuses easily to the target tissues through cellular membranes and is translocated to the RARs through CRABP (Cellular Retinoic Acid Binding Protein) (Ref.1 & 2).

The mechanism of all-trans-Retinoic Acid-induced Apoptosis is through Mitochondrial Dysfunction involving TRAIL (TNF-Related Apoptosis-Inducing Ligand) and it’s Death Receptors, the TRAILRs (TNF-Related Apoptosis-Inducing Ligand Receptors). all-trans-Retinoic Acid activate Ifns (Interferons) and both function synergistically to activate TRAILRs and Caspase8 (Cysteine Aspartate Specific Protease-8) that in turn induce the mitochondrial damage leading to the release of CytoC (Cytochrome-C). The TRAILRs contain the functional DDs (Death Domains), capable of inducing Apoptosis. Binding of TRAIL to TRAILRs and subsequent all-trans-Retinoic Acid-mediated activation leads to the recruitment of the Apoptosis Regulator FADD (Fas-Associated via Death Domain), which functions as a molecular bridge to Caspase8. Upon activation the TRAILRs indirectly bind to FADD via the GTP-binding protein DAP3 (Death-Associated Protein-3). Caspase8 cleaves BID (BH3 Interacting Domain Death Agonist) and the tBID (Truncated BID) translocates to mitochondria, inducing CytoC release. CytoC in association with APAF1 (Apoptotic Protease Activating Factor-1) activates Caspase9 (Apoptosis Related Cysteine Protease-9). Caspase9, in turn, causes the cleavage of proteins required for cellular viability, resulting in Apoptosis. Caspase9 also activates Caspase3 which directly cleaves downstream substrates like PARPs (Poly (ADP-Ribose) Polymerases) (Ref.3). Apoptosis by TRAIL and TRAILRs is controlled by FLIP (FLICE Inhibitory Protein), which inhibits the activation of Caspase8. Another mechanism of all-trans-Retinoic Acid induced Apoptosis requires Cytokine-mediated stimulation of PLA2 activity, resulting in the generation of excess Arachidonic Acid and this pathway is chiefly functional in the brain cells. Retinoic Acid functions as an important regulatory signaling molecule for Cell Growth, Differentiation and Neurodegeneration both during embryogenesis and in adult stage. Retinoic Acid induced Apoptosis through Death Receptors is a potentially promising approach for treatments of diseases like Schizophrenia, Alzheimer Disease and also for Cancer therapy (Ref.4 & 5).

References:

1. Lee GS, Kochhar DM, Collins MD.
Retinoid-induced limb malformations.
Curr. Pharm. Des. 2004;10(22):2657-99.
PubMed ID: 15320736

2. Miano JM, Berk BC.
Retinoids: versatile biological response modifiers of vascular smooth muscle phenotype.
Circ. Res. 2000 Sep 1;87(5):355-62.
PubMed ID: 10969032

3. Farooqui AA, Antony P, Ong WY, Horrocks LA, Freysz L.
Retinoic acid-mediated phospholipase A2 signaling in the nucleus.
Brain. Res. Brain Res. Rev. 2004 Jul;45(3):179-95.
PubMed ID: 15210303

4. Gottlieb RA.
Programmed cell death.
Drug. News Perspect. 2000 Oct;13(8):471-6.
PubMed ID: 12937619

5. Quadro L, Hamberger L, Gottesman ME, Wang F, Colantuoni V, Blaner WS, Mendelsohn CL.
Pathways of vitamin A delivery to the embryo: insights from a new tunable model of embryonic vitamin A deficiency.
Endocrinology. 2005 Jun 30.
PubMed ID: 15994349



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